Functional genomics has enabled high-throughput methods for identifying bacterial genes and proteins that are differentially expressed in response to host defenses. In particular, both microarrays and high throughput proteomics have been used to identify bacterial genes associated with resistance to host defenses. In addition to microarrays and proteomics, other high-throughput methods have been used to identify bacterial genes upregulated in response to phagocytosis, including differential fluorescence induction (DFI), random luciferase transcriptional fusions, and selective capture of transcribed sequences (SCOTS).
However, genes that are differentially regulated in response to a host defense are not necessarily the same as those that are required for survival. For example, not all of the genes that have increased expression following phagocytosis are required for survival in phagocytes. It is also possible that not all of the genes required for survival have enough change in expression to allow detection. Therefore, mutagenesis studies complement gene and protein expression studies and are likely to detect a unique set of genes that are required for survival.
A major hurdle in identifying bacterial mutants susceptible to host defenses is that the screening methods tend to be labor intensive. Fields et al. identified 83 S. typhimurium transposon mutants with impaired macrophage survival by screening individual transposon mutants with phagocytes in 96-well plates (Fields, P. I., Swanson, R. V., Haidaris, C. G. & Heffron, F. Mutants of Salmonella typhimurium that cannot survive within the macrophage are avirulent. Proc Natl Acad Sci USA 83, 5189-5193 (1986)). However, this assay required bacterial quantification from each well by plate counts. Zhao et al. used the same method to identify 37 Salmonella mutants susceptible to chicken macrophages (Zhao, Y. et al. Identification of genes affecting Salmonella enterica serovar Enteritidis infection of chicken macrophages. Infect Immun 70, 5319-5321 (2002)).
Improvements to allow high-throughput mutant screening have been reported, including a method for screening of bacterial mutants using bioluminescence to identify mycobacterial genes required for survival in macrophages and a microarray-based method for screening mutants. However, none of the previously described methods allow monitoring of bacterial mutant viability at multiple time points. In addition, previous bioluminescence based methods required a bacterial lysis step and addition of extraneous luciferin substrate and ATP for determining luciferase activity, which increases handling and cost while reducing the screening efficiency.
Edwardsiella ictaluri is the causative agent of enteric septicemia of catfish, an important disease of farm-raised channel catfish. Like some other species in the Enterobacteriaceae, E. ictaluri has the ability to resist killing by professional phagocytes. In particular, E. ictaluri is resistant to channel catfish neutrophils, which is an important aspect of pathogenesis because neutrophils are the predominant cell type in channel catfish intestinal tract immune cells. The intestine is an important site of entry for E. ictaluri. E. ictaluri is also resistant to killing by the alternative complement pathway in channel catfish.
There exists a need for a high throughput method for screening bacterial mutants to be used in live attenuated vaccines, such as one against Edwardsiella ictaluri in catfish.